SUMMARY Detection and treatment of glaucoma would benefit greatly from a thorough understanding of the mechanisms leading to neural tissue degeneration, and the development of a technique to evaluate eye-specific susceptibility to damage before it occurs. Our long-term goal is the development of such a technique. Neural tissue degeneration in early glaucoma is often localized to specific regions of the optic nerve head (ONH), and aging and elevated intraocular pressure (IOP) increase the risk. Our central hypothesis is that the architecture of the connective tissues of the ONH, and in particular of the lamina cribrosa (LC) within, determines the local robustness and sensitivity to IOP, and with this the regional susceptibility to neural tissue damage in early glaucoma, at all levels of IOP. In the previous project period, we used imaging tools based on polarized light microscopy (PLM) to obtain micron-scale information of the tissues of the ONH, including maps of collagen fiber alignment and the degree of stretch or relaxation of the fibers, referred to as crimp. We identified patterns in the crimp within the ONH and around the globe, and an age-related decrease crimp. However, because of the lack of suitable technology, analysis of the sensitivity to IOP was limited to comparing eyes fixed at different IOPs. We used modeling to predict effects of architecture on tissue properties and sensitivity to IOP, but, again, the lack of experimental tools made it impossible to actually test the relationship. We have developed two state-of- the-art imaging techniques, SPLM and IPOL. SPLM provides PLM-like data, from fresh thick ONH tissues. Coupled with optical coherence tomography, it provides excellent details of the ONH sensitivity to IOP during inflation. IPOL improves speed, resolution and sensitivity, resolving in real time, not just collagen bundles, but the details of the fibers forming the bundles. Using IPOL in a novel micro-mechanical testing system reveals fine tissue details while under controlled load, thus allowing direct measurement of local mechanical properties. Finally, we have developed a fiber-based simulation technique that allows modeling the tissues in a highly realistic way. We will use these techniques to measure, in Aim 1, the ONH biomechanical sensitivity to IOP and test the hypothesis that regions of known susceptibility to damage in early glaucoma are more sensitive to IOP. We test the hypothesis that age is associated with lower sensitivity to IOP in the PPS and higher in the LC. In Aim 2 we test the prediction that age is associated with changes in the architecture of the tissues of the ONH at multiple scales. In Aim 3, we will measure directly the mechanical properties of the tissues, and use modeling to test the hypothesis that the changes in sensitivity to IOP and biomechanical properties with age can be accounted for by the changes in microstructure. This project will answer both novel and long-standing question on the roles of architecture, IOP and aging on the mechanical insult to the neural tissues of the ONH, and the causes underlying the patterns of tissue loss in glaucoma. This is an important step towards the ultimate goal of diagnosing eyes at risk of glaucoma at all ages and levels of IOP.